As a component of heme and a constituent of many iron-dependent enzymes, iron is essential for oxidative phosphorylation, erythropoiesis, and many other chemical reactions that are vital for survival of almost all living organisms. Iron deficiency afflicts 30% of the world's population, and can cause anemia, developmental retardation, fertility problems and immune dysfunction. However, iron is a double-edged sword. It can also be toxic or even lethal when present in excess. Although it has long been appreciated that body iron content and plasma iron levels are meticulously regulated, many of the molecular details of iron homeostasis are obscure, and even the basic question about how iron deficiency or excess are sensed remains elusive. Recently we used positional cloning to identify a protein with non-redundant function in the detection of iron deficiency. This work was aimed at the understanding of mask, an ENU-induced phenotype characterized by progressive alopecia and microcytic anemia, and iron deficiency in mice. Mask phenotype was traced to a splicing error affecting the type II transmembrane serine protease 6 (TMPRSS6). Moreover, our data revealed that TMPRSS6 is an essential component of the "low iron" sensing apparatus in hepatocytes. Specifically: 1. the normal function of TMPRSS6 is to inhibit the expression of hepcidin, the central regulator of iron homeostasis;2. the suppression of Hamp, the gene encoding hepcidin by TMPRSS6, dominates over all known stimulus-induced pathways for upregulation of hepcidin;3. the inhibitory effect of TMPRSS6 is mediated by proximal element(s) of the Hamp promoter;4. the proteolytic activity of TMPRSS6 is essential for Hamp suppression;5. the cytoplasmic domain of TMPRSS6 is able to autonomously drive the suppressive signal transduction. In addition, we investigated the transcription profiles in HepG2 cells (a liver carcinoma cell line) with TMPRSS6 overexpression under basal condition and upon stimulation with the potent upregulator of hepcidin expression, BMP2. A few genes were found to be differentially modulated by TMPRSS6 overexpression, providing possible links in the analysis of signal transduction. In this proposal, we plan to carry our work forward to fully characterize the signaling cascade emanating from TMPRSS6 in response to iron attenuation. Three courses of experimentation will be pursued. We will: 1. investigate the structural characteristics of TMPRSS6, hoping to gain insight on the proximal events in signal transduction initiated by TMPRSS6 on the cell membrane;2. perform affinity-based protein interaction studies, aiming to isolate the protein(s) that associate with TMPRSS6 in the signaling pathway;3. conduct a forward genetic screen to search for mutations that disrupt systemic iron balance in mice, in order to expand and complete our knowledge of iron homeostasis. Through these studies, we expect that we will not only be able to fully elucidate iron homeostatic regulation, but also create effective strategies combating various disorders of iron absorption. PUBLIC HEALTH RELEVANCE: This proposal is aimed at characterization of a signaling pathway that permits the detection of iron deficiency in the body, and explores the possibility of finding the complete set of regulatory proteins for iron homeostasis by a genetic approach.